Professor Lee Smith

A complex milieu of hormones initiates diverse signaling pathways within and between cells of the testis to support the processes of steroidogenesis, spermatogenesis, and ultimately male fertility. In this chapter, we discuss the roles, mechanisms, and impacts of hormone signaling within the testis. We describe the mechanisms by which luteinizing hormone promotes the production of testosterone, a hormone essential for spermatogenesis. We use information obtained from cell-specific knockout studies of the androgen receptor as well as cellular and gene expression studies to describe the multifaceted actions of testosterone within the testis. We examine the multiple signaling pathways regulated by follicle-stimulating hormone in Sertoli cells and discuss how these pathways interface with testosterone signaling. In addition, we review how thyroid hormone regulates the proliferation of Sertoli cells and thus determines the capacity for sperm production. Furthermore, the paracrine functions of testis-derived hormones, including estrogen as well as activin and inhibin, are discussed. Finally, the contributions of other hormones that act to fine-tune testicular functions in response to environmental inputs are discussed. Together, these combined data paint a vivid picture of a complex, hormone-regulated signaling environment in the testis that acts to ensure that correct testicular function is maintained throughout life.

Sperm develop from puberty in the seminiferous tubules, inside the blood-testis barrier to prevent their recognition as ¿non-self¿ by the immune system, and it is widely assumed that human sperm-specific proteins cannot access the circulatory or immune systems. Sperm-specific proteins aberrantly expressed in cancer, known as cancer-testis antigens (CTAs), are often pursued as cancer biomarkers and therapeutic targets based on the assumption they are neoantigens absent from the circulation in healthy men. Here, we identify a wide range of germ cell-derived and sperm-specific proteins, including multiple CTAs, that are selectively deposited by the Sertoli cells of the adult mouse and human seminiferous tubules into testicular interstitial fluid (TIF) that is ¿outside¿ the blood-testis barrier. From TIF, the proteins can access the circulatory- and immune systems. Disruption of spermatogenesis decreases the abundance of these proteins in mouse TIF, and a sperm-specific CTA is significantly decreased in TIF from infertile men, suggesting that exposure of certain CTAs to the immune system could depend on fertility status. The results provide a rationale for the development of blood-based tests useful in the management of male infertility and indicate CTA candidates for cancer immunotherapy and biomarker development that could show sex-specific and male-fertility-related responses.

The epididymis is an androgen-responsive organ, whose structure and functions are modulated by the coordination between androgen and epididymal cues. Highly regulated molecular interaction within the epididymis is required to support viable sperm development necessary for subsequent fertilization. In the present study, we extended our earlier findings on a promising epididymal protein, quiescin sulfhydryl oxidase 2 (QSOX2), and demonstrated a positive correlation between testosterone and QSOX2 protein synthesis through the use of loss- and restore-of-function animal models. Moreover, based on transcriptomic analyses and 2D culture system, we determined that an additional polarized effect of glutamate is indispensable for the regulatory action of testosterone on QSOX2 synthesis. In conclusion, we propose noncanonical testosterone signaling supports epididymal QSOX2 protein synthesis, providing a novel perspective on the regulation of sperm maturation within the epididymis.

Focal dysgenesis is a consistent feature of testicular dysgenesis syndrome (TDS) in humans. Rodent studies show that perturbation of androgens (e.g. following phthalate exposure) during a fetal masculinisation programming window (MPW) predisposes to a TDS phenotype. This study aimed to determine whether dissociation and reconstitution of rat fetal testis tissue during the MPW can be used to model and manipulate seminiferous cord development, including induction of focal dysgenesis, as described in TDS. Dissociated fetal rat testes were xenotransplanted subcutaneously into recipient mice for 4¿weeks. Transplanted mice were treated with vehicle or di-n-butyl-phthalate (DBP, a plasticising chemical known to induce testicular dysgenesis in vivo in rats). Testosterone production by the transplants was measured in recipient mice and immunofluorescence was performed on the retrieved transplants to identify features consistent with focal testicular dysgenesis. Re-aggregation of rat fetal testis tissue xenotransplants during the MPW results in reconstitution of seminiferous cords. Features of focal testicular dysgenesis were present in re-aggregated testis, including ectopic Sertoli cells and intratubular Leydig cells (ITLCs). DBP exposure of recipient mice reduced androgen-dependent seminal vesicle weight (8.3 vs 26.7¿mg; p < 0.05), but did not enhance features of focal dysgenesis including number of ITLCs (0.07 vs 0.10 cells/mm2; p > 0.05). We conclude that¿seminiferous cord reformation during the MPW results in development of focal dysgenesis. The system may be used to separate specific effects (e.g. androgen suppression) of individual chemical exposures from other mechanisms that may be conserved in TDS.

The developing male reproductive system may be sensitive to disruption by a wide range of exogenous ¿endocrine disruptors¿. In-utero exposure to environmental chemicals and pharmaceuticals have been hypothesized to have an impact in the increasing incidence of male reproductive disorders. The vulnerability to adverse effects as a consequence of such exposures is elevated during a specific ¿window of susceptibility¿ in fetal life referred to as the masculinisation programing window (MPW). Exposures that occur during prepuberty, such as chemotherapy treatment for cancer during childhood, may also affect future fertility. Much of our current knowledge about fetal and early postnatal human testicular development derives from studies conducted in animal models predictive for humans. Therefore, over recent years, testicular transplantation has been employed as a ¿direct¿ approach to understand the development of human fetal and prepubertal testis in health and disease. In this review we describe the potential use of human testis xenotransplantation to study testicular development and its application for (i) assessing the effects of environmental exposures in humans, and (ii) establishing fertility preservation options for prepubertal boys with cancer.

Testicular germ cell cancer develops from premalignant intratubular germ cell neoplasia, unclassified cells that are believed to arise from failure of normal maturation of fetal germ cells from gonocytes (OCT4 + /MAGEA4 - ) into pre-spermatogonia (OCT4 - /MAGEA4 + ). Intratubular germ cell neoplasia cell subpopulations based on stage of germ cell differentiation have been described, however the importance of these subpopulations in terms of invasive potential has not been reported. We hypothesized that cells expressing an immature (OCT4 + /MAGEA4 - ) germ cell profile would exhibit an increased proliferation rate compared with those with a mature profile (OCT4 + /MAGEA4 + ). Therefore, we performed triple immunofluorescence and stereology to quantify the different intratubular germ cell neoplasia cell subpopulations, based on expression of germ cell (OCT4, PLAP, AP2¿, MAGEA4, VASA) and proliferation (Ki67) markers, in testis sections from patients with preinvasive disease, seminoma, and non-seminoma. We compared these subpopulations with normal human fetal testis and with seminoma cells. Heterogeneity of protein expression was demonstrated in intratubular germ cell neoplasia cells with respect to gonocyte and spermatogonial markers. It included an embryonic/fetal germ cell subpopulation lacking expression of the definitive intratubular germ cell neoplasia marker OCT4, that did not correspond to a physiological (fetal) germ cell subpopulation. OCT4 + /MAGEA4 - cells showed a significantly increased rate of proliferation compared with the OCT4 + /MAGEA4 + population (12.8 versus 3.4%, P < 0.0001) irrespective of histological tumor type, reflected in the predominance of OCT4 + /MAGEA4 - cells in the invasive tumor component. Surprisingly, OCT4 + /MAGEA4 - cells in patients with preinvasive disease showed significantly higher proliferation compared to those with seminoma or non-seminoma (18.1 versus 10.2 versus 7.2%, P < 0.05, respectively). In conclusion, this study has demonstrated that OCT4 + /MAGEA4 - cells are the most frequent and most proliferative cell population in tubules containing intratubular germ cell neoplasia, which appears to be an important factor in determining invasive potential of intratubular germ cell neoplasia to seminomas.Modern Pathology advance online publication, 24 January 2014; doi:10.1038/modpathol.2013.246.

Background: Androgen signalling is essential both for male development and function of the male reproductive system in adulthood. Within the adult testis, Germ cells (GC) do not express androgen receptor (AR) suggesting androgen-mediated promotion of spermatogenesis must act via AR-expressing somatic cell-types. Several recent studies have exploited the Cre/lox system of conditional gene-targeting to ablate AR function from key somatic cell-types in order to establish the cell-specific role of AR in promotion of male fertility. In this study, we have used a similar approach to specifically ablate AR-signalling from Vascular Endothelial (VE) cells, with a view to defining the significance of androgen signalling within this cell-type on spermatogenesis. Findings. AR expression in VE cells of the testicular vasculature was confirmed using an antibody against AR. A Cre-inducible fluorescent reporter line was used to empirically establish the utility of a mouse line expressing Cre Recombinase driven by the Tie2-Promoter, for targeting VE cells. Immunofluorescent detection revealed expression of YFP (and therefore Cre Recombinase function) limited to VE cells and an interstitial population of cells, believed to be macrophages, that did not express AR. Mating of Tie2-Cre males to females carrying a floxed AR gene produced Vascular Endothelial Androgen Receptor Knockout (VEARKO) mice and littermate controls. Ablation of AR from all VE cells was confirmed; however, no significant differences in bodyweight or reproductive tissue weights could be detected in VEARKO animals and spermatogenesis and fertility was unaffected. Conclusions: We demonstrate the successful generation and empirical validation of a cell-specific knockout of AR from VE cells, and conclude that AR expression in VE cells is not essential for spermatogenesis or male fertility. © 2012 O'Hara et al.

BACKGROUND Androgens and paracrine signaling from mesenchyme/stroma regulate development and disease of the prostate, and gene profiling studies of inductive prostate mesenchyme have identified candidate molecules such as pleiotrophin (Ptn). METHODS Ptn transcripts and protein were localized by in situ and immunohistochemistry and Ptn mRNA was quantitated by Northern blot and qRT-PCR. Ptn function was examined by addition of hPTN protein to rat ventral prostate organ cultures, primary human fetal prostate fibroblasts, prostate cancer associated fibroblasts, and BPH1 epithelia. RESULTS During development, Ptn transcripts and protein were expressed in ventral mesenchymal pad (VMP) and prostatic mesenchyme. Ptn was localized to mesenchyme surrounding ductal epithelial tips undergoing branching morphogenesis, and was located on the surface of epithelia. hPTN protein stimulated branching morphogenesis and stromal and epithelial proliferation, when added to rat VP cultures, and also stimulated growth of fetal human prostate fibroblasts, prostate cancer associated fibroblasts, and BPH1 epithelia. PTN mRNA was enriched in patient-matched normal prostate fibroblasts versus prostate cancer associated fibroblasts. PTN also showed male enriched expression in fetal human male urethra versus female, and between wt male and ARKO male mice. Transcripts for PTN were upregulated by testosterone in fetal human prostate fibroblasts and organ cultures of female rat VMP. Ptn protein was increased by testosterone in organ cultures of female rat VMP and in rat male urethra compared to female. CONCLUSIONS Our data suggest that in the prostate Ptn functions as a regulator of both mesenchymal and epithelial proliferation, and that androgens regulate Ptn levels. © 2010 Wiley-Liss, Inc.

Over the past 20 years, genetic manipulation has revolutionised our understanding of male reproductive development and function. The advent of transgenic mouse lines has permitted elegant dissection of previously intractable issues. The development of the Cre/Lox system, which has permitted spatial and temporal localisation of genetic manipulation, has expanded upon this, and now makes up one of the primary approaches underpinning our increasing understanding of testis development and function. The success of conditional gene targeting is largely reliant upon the choice of Cre recombinase expressing mouse line, which is required to specifically target the correct cell type at the correct time. Presupposition that Cre lines will behave as expected has been one of the main oversights in the design of Cre/Lox experiments, as in practice, many Cre lines are prone to ectopic expression (both temporal and spatial), transgene silencing or genetic background effects. Empirical validation of the spatiotemporal profile of Cre expression prior to undertaking conditional gene targeting studies is essential and can be achieved through a combination of molecular and immunohistochemical approaches, along with in vivo examination of reporter gene expression in targeted tissues. This paper details the key considerations associated with exploitation of the Cre/Lox system and highlights a variety of validated Cre lines that have utility for conditional gene targeting within the testis. © 2011 Society for Reproduction and Fertility.

Despite the increasing interest in other classes of small RNAs, microRNAs (miRNAs) remain the most widely investigated and have been shown to play a role in a number of different processes in mammals. Many studies investigating miRNA function focus on the processing enzyme Dicer1, which is an RNAseIII protein essential for the biogenesis of active miRNAs through its cleavage of precursor RNA molecules. General deletion of Dicer1 in the mouse confirms that miRNAs are essential for development because embryos lacking Dicer1 fail to reach the end of gastrulation. Here we investigate the role of Dicer1 in urogenital tract development. We utilised a conditional allele of the Dicer1 gene and two Cre-expressing lines, driven by HoxB7 and Amhr2, to investigate the effect of Dicer1 deletion on both male and female reproductive tract development. Data presented here highlight an essential role for Dicer1 in the correct morphogenesis and function of the female reproductive tract and confirm recent findings that suggest Dicer1 is required for female fertility. In addition, HoxB7:Cre-mediated deletion in ureteric bud derivatives leads to a spectrum of anomalies in both males and females, including hydronephrotic kidneys and kidney parenchymal cysts. Male reproductive tract development, however, remains largely unaffected in the absence of Dicer1. Thus, Dicer1 is required for development of the female reproductive tract and also normal kidney morphogenesis. © 2009 Springer Science+Business Media, LLC.

The mammalian gonad arises as a bipotential primordium from which a testis or ovary develops depending on the chromosomal sex of the individual. We have previously used DNA microarrays to screen for novel genes controlling the developmental fate of the indifferent embryonic mouse gonad. Maestro (Mro), which encodes a HEAT-repeat protein, was originally identified as a gene exhibiting sexually dimorphic expression during mouse gonad development. Wholemount in situ hybridisation analysis revealed Mro to be expressed in the embryonic male gonad from approximately 11.5 days post coitum, prior to overt sexual differentiation. No significant expression was detected in female gonads at the same developmental stage. In order to address its physiological function, we have generated mice lacking Maestro using gene targeting. Male and female mice homozygous for a Mro null allele are viable and fertile. We examined gonad development in homozygous male embryos in detail and observed no differences when compared to wild-type controls. Immunohistochemical analysis of homozygous mutant testes of adult mice revealed no overt abnormalities. Expression profiling using DNA microarrays also indicated no significant differences between homozygote embryonic male gonads and controls. We conclude that Maestro is dispensable for normal male sexual development and fertility in laboratory mice; however, the Mro locus itself does have utility as a site for insertion of transgenes for future studies in the fields of sexual development and Sertoli cell function. © 2008 Smith et al.

Background. To date, the earliest stage at which the orientation of the anterior-posterior axis in the mouse embryo is distinguishable by asymmetric gene expression is shortly after E5.5. At E5.5, prospective anterior markers are expressed at the distal tip of the embryo, whereas prospective posterior markers are expressed more proximally, close to the boundary with the extraembryonic region. Results. To contribute to elucidating the mechanisms underlying the events involved in early patterning of the mouse embryo, we have carried out a microarray screen to identify novel genes that are differentially expressed between the distal and proximal parts of the E5.5 embryo. Secondary screening of resulting candidates by in situ hybridisation at E5.5 and E6.5 revealed novel expression patterns for known and previously uncharacterised genes, including Peg10, Ctsz1, Cubilin, Jarid1b, Ndrg1, Sfmbt2, Gjb5, Talia and Plet1. The previously undescribed gene Talia and recently identified Plet1 are expressed specifically in the distal-most part of the extraembryonic ectoderm, adjacent to the epiblast, and are therefore potential candidates for regulating early patterning events. Talia and the previously described gene XE7 define a gene family highly conserved among metazoans and with a predicted protein structure suggestive of a post-transcriptional regulative function, whilst Plet1 appears to be mammal-specific and of unknown function. Conclusion. Our approach has allowed us to compare expression between dissected parts of the egg cylinder and has identified multiple genes with novel expression patterns at this developmental stage. These genes are potential candidates for regulating tissue interactions following implantation. © 2007 Frankenberg et al; licensee BioMed Central Ltd.

Comparative genome hybridization (CGH) to DNA microarrays (array CGH) is a technique capable of detecting deletions and duplications in genomes at high resolution. However, array CGH studies of the human genome noting false negative and false positive results using large insert clones as probes have raised important concerns regarding the suitability of this approach for clinical diagnostic applications. Here, we adapt the Smith-Waterman dynamic-programming algorithm to provide a sensitive and robust analytic approach (SW-ARRAY) for detecting copy-number changes in array CGH data. In a blind series of hybridizations to arrays consisting of the entire tiling path for the terminal 2 Mb of human chromosome 16p, the method identified all monosomies between 267 and 1567 kb with a high degree of statistical significance and accurately located the boundaries of deletions in the range 267-1052 kb. The approach is unique in offering both a nonparametric segmentation procedure and a nonparametric test of significance. It is scalable and well-suited to high resolution whole genome array CGH studies that use array probes derived from large insert clones as well as PCR products and oligonucleotides. © The Author 2005. Published by Oxford University Press. All rights reserved.

Gene expression is a central concept in molecular biology: its control, frequently exquisite in terms of cell specificity and timing, forms part of our explanation of most biological processes. The importance of the control of gene expression for developmental biologists is made obvious by just considering the nature of their discipline. Development is the term we use to describe the coordination in time and space of numerous cellular activities such as mitosis, migration, differentiation and apoptosis. Understanding the role of genes in these processes thus necessitates the use of methods to determine patterns of transcription during development with a high degree of sensitivity and specificity, conventionally by in situ hybridization. However, there is a widespread conviction amongst biologists that the description of gene expression patterns is of no immediate functional relevance: definitive functional data are the exclusive prerogative of biochemistry and genetics. In this review of recent applications of DNA microarray technology by developmental biologists, we suggest that genome-wide expression profiling has met with some resistance owing to such preconceived ideas about the status of gene expression pattern descriptions and, in particular, the format in which these are delivered by microarrays.

Mammalian sex determination depends on the presence or absence of SRY transcripts in the embryonic gonad. Expression of SRY initiates a pathway of gene expression resulting in testis development. Here, we describe a novel gene potentially functioning in this pathway using a cDNA microarray screen for genes exhibiting sexually dimorphic expression during murine gonad development. Maestro (Mro) transcripts are first detected in the developing male gonad before overt testis differentiation. By 12.5 days postcoitus (dpc), Mro transcription is restricted to the developing testis cords and its expression is not germ cell-dependent. No expression is observed in female gonads between 10.5 and 14.5 dpc. Maestro encodes a protein containing HEAT-like repeats that localizes to the nucleolus in cell transfection assays. Maestro maps to a region of mouse chromosome 18 containing a genetic modifier of XX sex reversal. We discuss the possible function of Maestro in light of these data. © 2003 Wiley-Liss, Inc.

The potential of expression analysis using cDNA microarrays to address complex problems in a wide variety of biological contexts is now being realised. A limiting factor in such analyses is often the amount of RNA required, usually tens of micrograms. To address this problem researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per mRNA molecule or increasing the amount of target available for labelling by use of an amplification procedure. We present a novel DNA-based method in which an oligonucleotide is incorporated into the 3' end of cDNA during second-strand cDNA synthesis. This sequence provides an annealing site for a single complementary heel primer that directs Taq DNA polymerase amplification of cDNA following multiple cycles of denaturation, annealing and extension. The utility of this technique for transcriptome-wide screening of relative expression levels was compared to two alternative methodologies for production of labelled cDNA target, namely incorporation of fluorescent nucleotides by reverse transcriptase or the Klenow fragment. Labelled targets from two distinct mouse tissues, adult liver and kidney, were compared by hybridisation to a set of cDNA microarrays containing 6500 mouse cDNA probes. Here we demonstrate, through a dilution series of cDNA derived from 10 micro g of total RNA, that it is possible to produce datasets comparable to those produced with unamplified targets with the equivalent of 30 ng of total RNA. The utility of this technique for microarray analysis in cases where sample is limited is discussed.

We report that Gata-2 is expressed in a sexually dimorphic fashion during mouse gonadogenesis. Gata-2 transcripts accumulate rapidly in the fetal ovary from 11.5 days post coitum (dpc) onwards, but are not detected in the fetal testis throughout the period studied (10.5-15.5 dpc). Ovarian expression of Gata-2 ceases by 15.5 dpc. Examination of ovaries from embryos homozygous for the extreme allele of c-kit(We) (Nature, 335, 88; Cell, 55, 185) demonstrates that ovarian Gata-2 expression is dependent upon the presence of germ cells. Comparative in situ hybridisation using the germ cell marker Oct4 (EMBO J., 8, 2543) indicates that Gata-2 transcripts are restricted to the germ cell lineage at 13.5 dpc. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

The characteristic curd of cauliflower (Brassica oleracea var. botrytis L.) consists of proliferating, arrested inflorescence and floral meristems. However, the origins and events leading to the domestication of this important crop trait remain unclear. A similar phenotype observed in the ap1-1/cal-1 mutant of Arabidopsis thaliana led to speculation that the orthologous genes from B. oleracea may be responsible for this characteristic trait. We have carried out a detailed molecular and genetic study, which allows us to present a genetic model based on segregation of recessive alleles at specific, mapped loci of the candidate genes BoCAL and BoAP1. This accounts for differences in stage of arrest between cauliflower and Calabrese broccoli (B. oleracea var. italica Plenck), and predicts the intermediate stages of arrest similar to those observed in Sicilian Purple types. Association of alleles of BoCAL-a with curding phenotypes of B. oleracea is also demonstrated through a survey of crop accessions. Strong correlations exist between specific alleles of BoCAL-a and discrete inflorescence morphologies. These complementary lines of evidence suggest that the cauliflower curd arose in southern Italy from a heading Calabrese broccoli via an intermediate Sicilian crop type. PCR-based assays for the two key loci contributing to curd development are suitable for adoption in marker-assisted selection.

The mammalian sex-determining pathway is controlled by the presence or absence of SRY expression in the embryonic gonad. Expression of SRY in males is believed to initiate a pathway of gene expression resulting in testis development. In the absence of SRY, ovary development ensues. Several genes have now been placed in this pathway but our understanding of it is far from complete and several functional classes of protein appear to be absent. Sex-determining genes frequently exhibit sexually dimorphic patterns of expression in the developing gonad both before and after overt differentiation of the testis or ovary. In order to identify additional sex-determining or gonadal differentiation genes we have examined gene expression in the developing gonads of the mouse using cDNA microarrays constructed from a normalized urogenital ridge library. We screened for genes exhibiting sexually dimorphic patterns of expression in the gonad at 12.5 and 13.5 days post-coitum, after overt gonad differentiation, by comparing complex cDNA probes derived from male and female gonadal tissue at these stages on microarrays. Using in situ hybridization analysis we show here that two genes identified by this screen, protease nexin-1 (Pn-1) and vanin-1 (Vnn1), exhibit male-specific expression prior to overt gonadal differentiation and are detected in the somatic portion of the developing gonad, suggesting a possible direct link to the testis-determining pathway for both genes.